1DE-MS profiling of rat cerebral cortex proteins of focal cerebral ischemia-reperfusion injury by combining SDS-PAGE, gel slicing, quantitative LC-MS/MS and profile reconstruction

A rat model of focal cerebral ischemia-reperfusion (I/R) injury was established by 2 hour-middle cerebral artery occlusion (MCAO) followed with 1, 7 and 14 days of reperfusion. Validation of the model was done by the measurement on cerebral blood flow, infarct volume, histological damage and neurological functions. The rat cerebral cortex proteins from the six animal groups (sham and I/R; 1D, 7D and 14D; 4 rats per group) were analyzed with an approach we termed as "1DE-MS profiling", by combining SDS-PAGE, gel slicing, quantitative LC-MS/MS and profile reconstruction. The samples were separated by gradient SDS-PAGE and each lane of the samples was cut into a set of 35 equal-sized (1.1 mm × 1.1 mm) square pieces. All the gel pieces were analyzed with standardized procedures of in-gel digestion and LC-MS/MS. A total of 5943 proteins (37 keratins included) were detected and the MS-detected abundance data were used to reconstruct the gel distributions in the six groups for all the proteins. All the 1DE-MS profiles were deposited in this dataset. The proteins were numbered alphabetically by their UniProtKB entry names, from #1 to #5943, as listed in "list of the 5943 proteins.xlsx". The data and the 1DE-MS profiles in two plot formats (color density and histogram) were provided in the six Excel files, each protein in a separate worksheet with the name "# protein -UniProtKB entry name" (e.g., 1536 -ALDOC_RAT). This dataset contains a significant amount of information of high complexity, which was presented visually by the 1DE-MS profiles. The observations included, 1) several hundreds of proteins were co-detected at any position in the gels, 2) about one-third of the proteins were detected at more than one positions or with broad distributions in the gels, 3) about one-fourth of the proteins were detected in the gels at the positions with the abundance-peak apparent molecular masses (Mws) obviously different from their calculated Mws, 4) the changes of a protein caused by different physiological or pathological conditions were not just about the total quantity, but of its structural information-rich profile. Examination of some of the profiles revealed the formation of the detected protein forms, often termed as "proteoforms" in recent years, involved alternative splicing, proteolytic processing, PTMs such as glycosylation, phosphorylation and ubiquitination, and degradation or fragmentation. We think this dataset would provide useful information for rat brain protein analysis and the method of 1DE-MS profiling would provide an option for proteomic analysis, from the view of proteoforms that concerns protein structures.本数据集通过2小时大脑中动脉闭塞（middle cerebral artery occlusion, MCAO）法构建局灶性脑缺血再灌注（focal cerebral ischemia-reperfusion, I/R）损伤大鼠模型，随后分别设置再灌注1天、7天及14天三个时间节点组。通过检测脑血流量、脑梗死体积、组织学损伤及神经功能对该模型进行验证。 本研究对6组大鼠（假手术组、缺血再灌注组；再灌注1天组、7天组及14天组，每组4只大鼠）的大脑皮层蛋白质组采用我们命名为‘一维凝胶电泳-质谱联用分析（1DE-MS profiling）’的方法进行分析，该方法整合了十二烷基硫酸钠聚丙烯酰胺凝胶电泳（sodium dodecyl sulfate polyacrylamide gel electrophoresis, SDS-PAGE）、凝胶切片、定量液相色谱-串联质谱（liquid chromatography-tandem mass spectrometry, LC-MS/MS）及图谱重构技术。样品通过梯度SDS-PAGE进行分离，每泳道的凝胶被切割为35块尺寸均一（1.1 mm × 1.1 mm）的方形凝胶块。所有凝胶块均采用标准化的胶内酶解及LC-MS/MS流程进行分析。最终共检测到5943种蛋白质（包含37种角蛋白），并利用质谱检测得到的丰度数据，重构了所有蛋白质在6组大鼠凝胶中的分布情况。本数据集完整收录了所有的1DE-MS分析图谱。所有蛋白质根据其UniProtKB条目名称按字母顺序编号，编号范围为#1至#5943，详细列表见"5943种蛋白质列表.xlsx"。本数据集以两种绘图格式（彩色密度图与直方图）提供了相关数据及1DE-MS图谱，所有内容存储于6个Excel文件中；每个蛋白质单独占用一个工作表，工作表命名格式为"#编号 - UniProtKB条目名称"（例如：1536 -ALDOC_RAT）。 本数据集包含大量高复杂度的信息，这些信息通过1DE-MS图谱以可视化形式呈现。本研究得到的主要观测结果包括：1）数百种蛋白质可在凝胶的任意位置共同被检测到；2）约三分之一的蛋白质可在凝胶的多个位置被检测到，或呈现较宽的分布范围；3）约四分之一的蛋白质在凝胶中的丰度峰值表观分子量（apparent molecular masses, Mws）与理论计算分子量（calculated Mws）存在显著差异；4）不同生理或病理条件下蛋白质的变化不仅体现为总表达量的改变，还与其结构信息丰富的图谱特征相关。对部分图谱的分析显示，检测到的蛋白质形式（近年来常被称为"蛋白质变体（proteoforms）"）的形成涉及可变剪接、蛋白水解加工、糖基化、磷酸化及泛素化等翻译后修饰（post-translational modifications, PTMs），以及蛋白质降解或片段化过程。从关注蛋白质结构的蛋白质变体研究视角出发，本数据集可为大鼠脑蛋白质组分析提供有价值的参考信息，而1DE-MS图谱分析方法也可为蛋白质组学研究提供一种新的技术选择。